Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

BACKGROUND AND PURPOSE

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca²⁺ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

EXPERIMENTAL APPROACH

The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca²⁺ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence.

KEY RESULTS

SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca²⁺ ([Ca²⁺]_i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na⁺/Ca²⁺ exchanger (NCX) with an EC₅₀ of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G₂ phases and activated p38-MAPK and JNK, which were all prevented by the Ca²⁺ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells.

CONCLUSIONS AND IMPLICATIONS

At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca²⁺]_i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca²⁺ homeostasis and suppress human glioblastoma cells.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Aim:

To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.

Methods:

Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na⁺/H⁺ exchanger 1 (NHE1) and calpain, intracellular free Ca²⁺level ([Ca²⁺]_i), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE^−/−) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.

Results:

LPS treatment increased NHE1 activity and [Ca²⁺]_i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca²⁺-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca²⁺]_i. and calpain activity. In the presence of the Ca²⁺ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE^−/− mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.

Conclusion:

LPS stimulates NHE1 activity, increases [Ca²⁺]_i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Omega-3 fatty acids induce Ca2+ mobilization responses in human colon epithelial cell lines endogenously expressing FFA4

Aim:

Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro.

Methods:

HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4.

Results:

Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca²⁺]_i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca²⁺]_i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca²⁺]_i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca²⁺ATPase inhibitor thapsigargin, but was not affected by G_i/o protein inhibitor PTX or IP₃R inhibitor 2-APB.

Conclusion:

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca²⁺ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive G_i/o protein, followed by Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and Ca²⁺ influx across the plasma membrane.     

Resveratrol protects rabbit ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca2+ overload

Aim:

To investigate whether resveratrol suppressed oxidative stress-induced arrhythmogenic activity and Ca²⁺ overload in ventricular myocytes and to explore the underlying mechanisms.

Methods:

Hydrogen peroxide (H₂O₂, 200 μmol/L)) was used to induce oxidative stress in rabbit ventricular myocytes. Cell shortening and calcium transients were simultaneously recorded to detect arrhythmogenic activity and to measure intracellular Ca²⁺ ([Ca²⁺]_i). Ca²⁺/calmodulin-dependent protein kinases II (CaMKII) activity was measured using a CaMKII kit or Western blotting analysis. Voltage-activated Na⁺ and Ca²⁺ currents were examined using whole-cell recording in myocytes.

Results:

H₂O₂ markedly prolonged Ca²⁺ transient duration (CaTD), and induced early afterdepolarization (EAD)-like and delayed afterdepolarization (DAD)-like arrhythmogenic activity in myocytes paced at 0.16 Hz or 0.5 Hz. Application of resveratrol (30 or 50 μmol/L) dose-dependently suppressed H₂O₂-induced EAD-like arrhythmogenic activity and attenuated CaTD prolongation. Co-treatment with resveratrol (50 μmol/L) effectively prevented both EAD-like and DAD-like arrhythmogenic activity induced by H₂O₂. In addition, resveratrol markedly blunted H₂O₂-induced diastolic [Ca²⁺]_i accumulation and prevented the myocytes from developing hypercontracture. In whole-cell recording studies, H₂O₂ significantly enhanced the late Na⁺ current (I_Na,L) and L-type Ca²⁺ current (I_Ca,L) in myocytes, which were dramatically suppressed or prevented by resveratrol. Furthermore, H₂O₂-induced ROS production and CaMKII activation were significantly prevented by resveratrol.

Conclusion:

Resveratrol protects ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca²⁺ overload through inhibition of I_Na,L/I_Ca,L, reduction of ROS generation, and prevention of CaMKII activation.     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

Aim:

To identify the mechanisms underlying the elevation of intracellular Ca²⁺ level ([Ca²⁺]_i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons.

Methods:

Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca²⁺]_i was measured using fura-2 AM. Ca²⁺ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay.

Results:

Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca²⁺]_i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons depended on extracellular Ca²⁺, and was blocked by P/Q-type Ca²⁺channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca²⁺ channel blocker nifedipine (10 μmol/L) or N-type Ca²⁺channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca²⁺ current in ARC neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L).

Conclusion:

Lowering glucose level enhances the activity of P/Q type Ca²⁺channels and elevates [Ca²⁺]_i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.     

cAMP- and Ca²(+) /calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

BACKGROUND AND PURPOSE

Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms.

EXPERIMENTAL APPROACH

Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca²⁺]_i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16.

KEY RESULTS

Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC₅₀: 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF₂ receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca²⁺]_i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca²⁺]_i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca²⁺]_i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93.

CONCLUSIONS AND IMPLICATIONS

Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF₂ receptors in a cAMP/PKA- and Ca²⁺/CaMKII-dependent manner. This enhancement was accompanied by Ca²⁺-dependent arrhythmogenic effects mediated by PKA and CaMKII.     

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose:

Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery.

Experimental approach:

The cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery.

Key results:

Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR₁) antagonist, while it was mimicked by PAR₁-activating peptide (PAR₁AP), but not PAR₄AP. The thrombin-induced contraction was associated with a small elevation of [Ca²⁺]_i and an increase in MLC20 phosphorylation. Thrombin and PAR₁AP induced a greater increase in tension for a given [Ca²⁺]_i elevation than that obtained with high K⁺-depolarization. They also induced a contraction at a fixed Ca²⁺ concentration in α-toxin-permeabilized preparations.

Conclusions and implications:

The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR₁ and thereby increasing the sensitivity of the myofilament to Ca²⁺. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.     

Mechanism of asynchronous Ca2+ waves underlying agonist-induced contraction in the rat basilar artery

Background and purpose:

Uridine 5''-triphosphate (UTP) is a potent vasoconstrictor of cerebral arteries and induces Ca²⁺ waves in vascular smooth muscle cells (VSMCs). This study aimed to determine the mechanisms underlying UTP-induced Ca²⁺ waves in VSMCs of the rat basilar artery.

Experimental approach:

Isometric force and intracellular Ca²⁺ ([Ca²⁺]_i) were measured in endothelium-denuded rat basilar artery using wire myography and confocal microscopy respectively.

Key results:

Uridine 5''-triphosphate (0.1–1000 µmol·L⁻¹) concentration-dependently induced tonic contraction (pEC₅₀ = 4.34 ± 0.13), associated with sustained repetitive oscillations in [Ca²⁺]_i propagating along the length of the VSMCs as asynchronized Ca²⁺ waves. Inhibition of Ca²⁺ reuptake in sarcoplasmic reticulum (SR) by cyclopiazonic acid abolished the Ca²⁺ waves and resulted in a dramatic drop in tonic contraction. Nifedipine reduced the frequency of Ca²⁺ waves by 40% and tonic contraction by 52%, and the nifedipine-insensitive component was abolished by SKF-96365, an inhibitor of receptor- and store-operated channels, and KB-R7943, an inhibitor of reverse-mode Na⁺/Ca²⁺ exchange. Ongoing Ca²⁺ waves and tonic contraction were also abolished after blockade of inositol-1,4,5-triphosphate-sensitive receptors by 2-aminoethoxydiphenylborate, but not by high concentrations of ryanodine or tetracaine. However, depletion of ryanodine-sensitive SR Ca²⁺ stores prior to UTP stimulation prevented Ca²⁺ waves.

Conclusions and implications:

Uridine 5''-triphosphate-induced Ca²⁺ waves may underlie tonic contraction and appear to be produced by repetitive cycles of regenerative Ca²⁺ release from the SR through inositol-1,4,5-triphosphate-sensitive receptors. Maintenance of Ca²⁺ waves requires SR Ca²⁺ reuptake from Ca²⁺ entry across the plasma membrane via L-type Ca²⁺ channels, receptor- and store-operated channels, and reverse-mode Na⁺/Ca²⁺ exchange.     

Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca2+]i in mesenteric microvessels of pregnant rats

Background and Purpose

Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ET_AR), but could activate ET_BR in the endothelium and release vasodilator substances. However, the roles of ET_BR in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear.

Experimental Approach

Pressurized mesenteric microvessels from pregnant and virgin Sprague–Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca²⁺]_i.

Key Results

High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [Ca²⁺]_i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca²⁺]_i. Pretreatment with the ET_BR antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [Ca²⁺]_i, and was more potent in pregnant than in virgin rats. ET-1 + ET_AR antagonist BQ-123, and the ET_BR agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [Ca²⁺]_i, suggesting up-regulated ET_BR-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor N_ω-nitro-l-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ET_BR in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ET_BR.

Conclusions and Implications

The enhanced ET_BR-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.